Heat-Killed Lacticaseibacillus paracasei GMNL-653 Exerts Antiosteoporotic Effects by Restoring the Gut Microbiota Dysbiosis in Ovariectomized Mice
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ORIGINAL RESEARCH
published: 04 February 2022
doi: 10.3389/fnut.2022.804210
Heat-Killed Lacticaseibacillus
paracasei GMNL-653 Exerts
Antiosteoporotic Effects by
Restoring the Gut Microbiota
Dysbiosis in Ovariectomized Mice
Jhih-Hua Jhong 1 , Wan-Hua Tsai 2 , Li-Chan Yang 3 , Chia-Hsuan Chou 2 , Tzong-Yi Lee 4,5 ,
Yao-Tsung Yeh 6,7 , Cheng-Hsieh Huang 6,8 and Yueh-Hsia Luo 9*
1
Department of Computer Science and Engineering, Yuan Ze University, Taoyuan, Taiwan, 2 Research and Development
Department, GenMont Biotech Incorporation, Tainan, Taiwan, 3 Department of Pharmacy, China Medical University, Taichung,
Taiwan, 4 School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China, 5 Warshel Institute for
Computational Biology, The Chinese University of Hong Kong, Shenzhen, China, 6 Aging and Diseases Prevention Research
Edited by: Center, Fooyin University, Kaohsiung, Taiwan, 7 Biomed Analysis Center, Fooyin University Hospital, Pingtung, Taiwan,
Sabina Górska, 8
Ph.D. Program in Environmental and Occupational Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan,
Polish Academy of Sciences, Poland 9
Department of Life Sciences, National Central University, Taoyuan, Taiwan
Reviewed by:
Guoju Hong,
University of Alberta, Canada Osteoporosis is a metabolic inflammatory disease, an imbalance occurs between
Giovanni Iolascon, bone resorption and formation, leading to bone loss. Anti-inflammatory diet is
University of Campania Luigi
considered having the potential to ameliorate osteoporosis. Heat-killed probiotics
Vanvitelli, Italy
Hailing Xin, exhibit health benefits in relation to their immunomodulatory effects, but the detail
Second Military Medical mechanism involved in gut microbiota balance, host metabolism, immunity, and bone
University, China
homeostasis remains unclear. In this study, we evaluated the antiosteoporotic effects of
*Correspondence:
Yueh-Hsia Luo heat-killed Lacticaseibacillus paracasei GMNL-653 in vitro and in ovariectomized (OVX)
yhluo@ncu.edu.tw mice. Furthermore, whole-genome sequencing and comparative genomics analysis
demonstrated potentially genes involved in antiosteoporotic activity. The GMNL-653
Specialty section:
This article was submitted to
exerts anti-inflammatory activity which restored gut microbiota dysbiosis and maintained
Nutritional Immunology, intestinal barrier integrity in the OVX mice. The levels of IL-17 and LPS in the sera
a section of the journal
decreased following GMNL-653 treatment compared with those of the vehicle control;
Frontiers in Nutrition
mRNA levels of RANKL were reduced and TGF-β and IL-10 enhanced in OVX-tibia tissue
Received: 29 October 2021
Accepted: 13 January 2022 after treatment. The levels of IL-17 were significantly associated with gut microbiota
Published: 04 February 2022 dysbiosis. Gut microbial metagenomes were further analyzed by PICRUSt functional
Citation: prediction, which reveal that GMNL-653 intervention influence in several host metabolic
Jhong JH, Tsai WH, Yang LC,
Chou CH, Lee TY, Yeh YT, Huang CH
pathways. The analysis of whole-genome sequencing accompanied by comparative
and Luo YH (2022) Heat-Killed genomics on three L. paracasei strains revealed a set of GMNL-653 genes that are
Lacticaseibacillus paracasei
potentially involved in antiosteoporotic activity. Our findings validated antiosteoporotic
GMNL-653 Exerts Antiosteoporotic
Effects by Restoring the Gut activity of heat-killed GMNL-653 using in vitro and in vivo models, to whole-genome
Microbiota Dysbiosis in sequencing and identifying genes potentially involved in this gut microbiota–bone axis.
Ovariectomized Mice.
Front. Nutr. 9:804210. Keywords: Lacticaseibacillus paracasei, gut microbiota, dysbiosis, whole-genome sequencing, gut-bone axis,
doi: 10.3389/fnut.2022.804210 IL-17A, RANKL (receptor activator for nuclear factor k B ligand), antiosteoporotic effects
Frontiers in Nutrition | www.frontiersin.org 1 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
INTRODUCTION one L. paracasei GMNL-653 strain reduced lipopolysaccharide
(LPS)-stimulated IL-6 and NO production in the mouse
Environmental factors influence host health through gut macrophage RAW264.7. Heat-killed GMNL-653 also increased
microbiota (1). Gut microbial communities can tune host calcium mineralization in human osteosarcoma MG63 cells
immunity, modulate gut endocrine function and neurological mineralizing in culture and protected the OVX mice from
signaling, and produce vital metabolites that influence the host bone loss, including increasing bone volume over tissue
(2). Gut dysbiosis is associated with many diseases, including volume (BV/TV) and bone mineral density (BMD). Our results
metabolic liver diseases, cardiometabolic diseases, obesity, and revealed that GMNL-653 restored ovariectomy-induced gut
type 2 diabetes (1). Moreover, studies and emerging evidence microbiota dysbiosis and maintained intestinal barrier integrity.
have indicated that dysregulated gut microbiota correlate with Additionally, in the OVX mice, GMNL-653 treatment reduced
decreased bone density, and inflammatory bowel disease (IBD) the IL-17 and LPS levels compared with those of the vehicle
(3–5). A close relationship between bone health and gut control group, reduced the mRNA levels of RANKL, and
microbiota exist that may involve in calcium absorption, bone increased the anti-inflammatory cytokines TGF-β and IL-10
mineralization, and immune signaling (6, 7). Osteoporosis, a in tibia tissue. We applied the whole-genome sequencing
common metabolic inflammatory disease, is characterized by low technique and comparative genomics analysis to further examine
bone density and the destruction of bone tissue; an imbalance these three L. paracasei. Our results indicated that the genes
occurs between bone resorption and formation, leading to related to carbohydrate transport/metabolism and the cell
bone loss. Osteoporosis-induced fractures of the femur and wall/membrane/envelope biogenesis of GMNL-653 are worthy of
vertebrae profoundly affect the life quality and mortality of aging future investigation. In summary, our results revealed that heat-
populations, representing a serious public health problem, and killed GMNL-653 exhibited antiosteoporotic activity through the
incurring high medical care costs. Postmenopausal osteoporosis gut microbiota–bone axis.
increases the expression of proinflammatory and osteogenic
cytokines, including interleukin 1 (IL-1), interleukin 6 (IL-
6), tumor necrosis factor-alpha (TNF-α), macrophage colony EXPERIMENTAL PROCEDURES
stimulating factor (MCSF), and the receptor activator of nuclear
factor kappa-light-chain-enhancer of activated B cells ligand
Bacterial Culture and Heat-Inactive
(RANKL) from osteoblasts, T cells, and B cells (8–11). Gut Preparations
dysbiosis not only induces local or systemic inflammation but Lactobacilli were cultured in 1 mL of MRS broth at 37◦ C for
also dysregulates nutrients and calcium across the intestine and 20 h, and subcultured at a 1:100 ratio in fresh MRS medium.
into systemic circulation. Several drugs (e.g., bisphosphonates, After 20 h of subculture, the bacteria were centrifuged at 13,000
denosumab, teriparatide, and abaloparatide) are used to treat rpm for 1 min, and the bacterial pellet was then washed twice
osteoporosis through the blocking of bone reabsorption, with PBS. The bacteria were suspended in PBS, with the
stimulating of bone formation, or both (12). Combined use of bacterial concentration adjusted to 1 × 1010 cells/mL. The
drugs and probiotic supplementation may represent a safe and heat-killing method was performed at 90◦ C for 30 min or
potentially effective therapy strategy for osteoporosis. through autoclaving at 121◦ C for 15 min. Lactobacilli used in
Although probiotics are considered non-pathogenic live this study were Limosilactobacillus reuteri (previously classified
microorganisms, the safety profile of live probiotic supplement as Lactobacillus reuteri; CCTCC M 209263), Lacticaseibacillus
remains unclear in certain circumstances, particularly in regard rhamnosus (CCTCC M 203098), Lacticaseibacillus salivarius
to the translocation of bacteria from intestinal tissue to systemic (GMNL-678), and five Lacticaseibacillus. paracasei strains
circulation (13). Hence, the utilization of nonviable heat-killed (#1:CCTCC M 204012; #2: CCTCC M 2011331; #3: GMNL-
probiotics has drawn increased attention. Several studies have 653/BCRC-910721; #4: GMNL-855; and #5: BCRC-16100/ATCC
reported that heat-killed probiotics exhibit health benefits in 11582) were provided by GenMont Biotech (Tainan, Taiwan).
terms of their immunomodulatory effect (14, 15) in in vitro and
in vivo models. Furthermore, inactivated Lactobacillus decreases Cell Culture
the production of IgE in an ovalbumin (OVA) mice model (16), The RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s
mediates the Th1/Th2 switch, and exhibits potential protective complete medium (DMEM) and supplemented with 10% fetal
effects in food allergies (17). The structural components of bovine serum (FBS; Invitrogen, MA, Unites States) and 20 µg/mL
probiotics, particularly the cell envelope, capsule, and cell gentamicin. The cells were seeded with 4 × 105 cells/well in a 24-
wall components, may play a role in these immunological well plate and then incubated in a humidified atmosphere at 37◦ C
effects (18, 19). However, the possible mechanisms underlying with 5% CO2 . The supernatant was removed, and the adhered
the influence of heat-killed probiotics on gut microbiota cells were washed twice with sterile PBS. Next, fresh DMEM
balance, host metabolism, immunity, and bone homeostasis without FBS was added for 2 h of starvation. The cells were
remain undetermined. treated with or without heat-killed bacteria for 2 h, and then LPS
In the present study, we investigated the antiosteoporotic (Escherichia coli O111:B4; SI-L4391, Sigma-Aldrich, St. Louis,
effects of three heat-killed Lacticaseibacillus paracasei (previously MO, United States) was added to yield a final concentration of
classified as Lactobacillus paracasei) strain GMNL-653 in vitro 100 ng/mL for a further 20 h of incubation. The supernatant was
and in ovariectomized (OVX) mice. First, we observed that collected for cytokine and NO measurement.
Frontiers in Nutrition | www.frontiersin.org 2 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
In vitro Model for Calcium Mineralization scanned. The locations of analysis were selected under proximal
Mineralization was induced on confluent monolayers to the growth plate 100 slices and excluded cortical bone.
through the addition of osteogenic culture medium (OIM)
containing 10% (v/v) FBS, 10 mM glycerol-phosphate, 100 mM
dexamethasone, and 0.05 mM vitamin C. Human osteosarcoma RNA Isolation and Quantitative Real-Time
osteoblastic MG63 cells were cultured in a 24-well plate (3 × Reverse Transcription-Polymerase Chain
104 cells/well) with DMEM containing 10% FBS. Heat-killed
GMNL-653 were adjusted to an appropriate density using OIM Reaction
medium, and MG63 cells were added. Alizarin red S (ARS) The tibia from the OVX and sham mice were dissected, and
staining was employed to evaluate calcium deposits. The cultures the soft tissue was removed. The sample was pulverized using
were incubated at 37◦ C with 5% CO2 with changes of OIM liquid nitrogen, and TRIzol reagent was added for RNA isolation.
medium every 7 days. After 28 days, the cells were washed twice The purified RNA concentration was measured and stored
with PBS and then fixed with 70% acetic acid for 1 h. The cells at −80◦ C, and cDNA was synthesized with the total RNA
were stained with 2% ARS (pH 4.1–4.5) at room temperature for (5 µg). Quantitative real-time reverse transcription-polymerase
30 min with gentle shaking. Following the removal of the ARS chain reaction (PCR) was used to measure TGF-β, RANKL,
dye, the cells were washed with H2 O five times. Excess water IL-10, bone morphogenetic proteins 2 (BMP2), tartrate-resistant
was removed from the plates, which were then stored at −20◦ C acid phosphatase 5b (Trap-5), and glyceraldehyde-3-phosphate
prior to dye extraction. For quantification of the ARS staining, dehydrogenase (GAPDH) expression, with the assays performed
200 µL of 10% (v/v) acetic acid was added to each well in the using 2× Rotor-Gene SYBR Green PCR Master Mix (204076;
24-well plate, and the plate was incubated at room temperature QIAGEN). The reaction mixtures were prepared by mixing
for 30 min with gentle shaking. The cells were then scraped from aliquots of cDNA, 3 µL of forward and reverse primer, and 5
the plate and transferred into a 1.5-mL microcentrifuge tube. µL of 2× Rotor-Gene SYBR Green PCR Master Mix to obtain
After being vortexed for 30 s, the tubes were heated to 85◦ C a final volume of 10 µL. The reaction mixtures were analyzed on
for 10 min and then placed into ice for 5 min. The tubes were a Rotor-Gene Q 2plex system. Quantitative values were obtained
centrifuged at 20,000 g for 15 min, and 200 µL of the supernatant from the threshold cycle (Ct) number. The relative mRNA levels
was transferred into a new 1.5-mL microcentrifuge tube. Next, 75 of the target genes were derived using the equation 2 – 1Ct,
µL of 10% (v/v) ammonium hydroxide was added to neutralize where 1Ct is Cttargetgene − CtGAPDH . Data were presented as the
the acid. Aliquots (50 µL) of the supernatant and standard were fold relative to the control value.
measured at 405 nm in a 96-well format.
16S Ribosomal RNA Gene Amplicon
Enzyme-Linked Immunosorbent Assay
Levels of IL-6 and interleukin 17 (IL-17A) were measured Sequencing
through enzyme-linked immunosorbent assay (BioLegend) Fecal samples were collected after 28 days of GMNL-653
according the instructions of the manufacturer. Sera LPS treatment, and all specimens were extracted using a QIAGEN
was detected using a Pierce LAL Chromogenic Endotoxin DNA kit according to the manufacturer’s instructions. DNA
Quantitation Kit (Thermo Scientific, MA, United State). samples were analyzed with a 260/280 OD in the range of
1.8 to 2.0. 16S ribosomal RNA (rRNA) PCR was performed
using metagenomic DNA as a template, which was amplified
Animals with the bacterial-specific primers S17 (5′ -TCG TCG GCA GCG
Twenty-four female ICR mice aged 8 to 10 weeks were housed TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC
in standard cages, maintained under specific pathogen-free WGC AG-3′ ) and A21 (5′ -GTC TCG TGG GCT CGG AGA
conditions, and fed sterilized food and autoclaved water. The TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA
mice were either OVX or sham-operated with an intraperitoneal ATC C-3′ ). The amplified DNA sizing was verified using a
injection of anesthetic following standard protocol and were fragment analyzer (5300; Agilent Technologies), and sequencing
divided into three categories with eight mice in each group was conducted using a Illumina MiSeq platform. DNA samples
(sham, OVX + H2 O, and OVX + GMNL-653). Mice in OVX were assigned indices and Illumina sequencing adapters with the
+ GMNL653 received GMNL-653 treatment once per day after Nextera XT Index Kit v2. After library construction, the samples
the OVX or sham surgery for 28 days. At the end of experiment were mixed using a 600-cycle MiSeq Reagent Kit v3 at a final
(4 weeks), the animals were sacrificed for further analysis. concentration of 4 pM, loaded onto a MiSeq cartridge, and then
transferred onto the instrument. Automated cluster generation
Micro-Computed Tomography Measures and a 2 × 300 bp paired-end sequencing run was performed.
The alterations in trabecular site of tibia were determined using The sequences generated passed through a filtering process to
a high resolution tomography image system micro-CT (SkyScan obtain the qualified reads. The total reads were merged, low-
1076, kontizh, Belgiumm resolution 18 µm per slice). After quality and chimera sequences were removed, and OTUs at a 97%
positioning right tibia, parameters such as total volume (TV, similarity with the Greengenes database (v13.8) were clustered.
mm3 ), bone volume/total volume (BV/TV, %), apparent density We employed the QIAGEN CLC Microbial Genomics Module
refer to bone mineral density (BMD) (mgHA·ccm-1) were (v10.1.1) for further analysis.
Frontiers in Nutrition | www.frontiersin.org 3 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
Processing and Statistical Analysis of instructions. After cluster generation, the library preparations
Metataxonomic Data were sequenced on an Illumina NovaSeq 6000 platform, and
The alpha diversity of taxonomic composition was measured paired-end reads were generated.
using the Shannon diversity index, which evaluates the overall
diversity of each group including the number of observed Sample Preparation and Sequencing on
species (richness) and the evenness of the observed taxonomic Oxford Nanopore Technologies GridION
composition. Beta diversity was measured using PCoA-Weighted DNA extraction was conducted in accordance with the
UniFrac to determine the difference in microbial composition EasyPure Genomic DNA Kit protocol (TransGen Biotech).
among groups. The OTU table was generated using the Library preparation was performed following Oxford Nanopore
QIAGEN CLC Microbial Genomics Module combined with Technologies (ONT) protocols in the form of native barcoding
linear discriminant analysis effect size (LEfSe). LEfSe was genomic DNA (with EXP-NBD103 and SQK-LSK108). Next,
performed using the Galaxy/Hutlab webtool to identify specific 400 ng of genomic DNA was fragmented with transposome
microbial markers among groups, with an 0.05 alpha value for randomly, with a barcode added at the same time. DNA
the factorial Kruskal–Wallis test and pairwise Wilcoxon test and was subsequently purified using AMPureXP beads and ligated
a linear discriminant analysis (LDA) score cutoff of 2.0. The using motor protein-complexed adapter to DNA ends. The
results revealed that the functional pathways had a significantly library was quantified using the Qubit Fluorometer, and the
different abundance at level 3 among the groups. Additionally, prepared library was loaded onto a running buffer-primed flow
Spearman’s correlation (calculated using the corrplot package cell for sequencing. Sequencing of the native genomic DNA
v0.84) and principal component analysis (PCA; performed using was performed on a single R9.4/FLO-MIN106 flow cell on
the ade4 package v1.7-16) were applied through the use of R the GridION.
language (v4.0.2). A comparison of the groups was undertaken
through two-tailed t-test analysis, and a p < 0.05 was considered Hybrid Assembly and Genome Annotation
statistically significant. We used GraphPad Prism 8 (GraphPad Genomes were constructed through a hybrid assembly method
Software, San Diego, CA, USA) to identify taxonomic differences of low-coverage ONT long-read (approximate mean of 6.2,
and generate relative abundance plots. 7.8, and 7.8 kbp for GMNL-653, GMNL-855, and BCRC-
16100, respectively), sequencing for generating scaffolds and
high-coverage Illumina short-read (550 bp) sequencing for
Library Preparation and Sequencing on the determining the consensus sequence. The de novo hybrid
Illumina Platform genome assembly approach was applied using the MaSuRCA
For the L. paracasei strain GMNL-653, library preparation was v3.3.1 assembler (20). Specifically, the MaSuRCA assembler first
conducted in accordance with the Illumina TruSeq Nano DNA reduces high-coverage Illumina reads to long super reads and
Sample Preparation Kit protocol. First, 200 ng of genomic DNA then aligns them with long ONT reads to generate even longer
was sonicated to approximately 550 bp. Fragmented DNA was super reads (21). Illumina short reads and ONT long reads were
processed to convert the overhangs into blunt ends, and the input into the MaSuRCA assembler without any preprocessing,
library size was selected using beads. After ends repair, a single as recommended in the MaSuRCA documentation. In addition,
“A” nucleotide was added to the 3′ ends of the blunt fragments, the MaSuRCA uses a high coverage of error-prone long
and the corresponding single “T” nucleotide on the adapter with reads generated by ONT to construct consensus sequences for
index was ligated to the fragments. The library size was verified regions not captured by the short Illumina reads. Consequently,
through electrophoresis, and the quantification was validated this approach combines the advantages of the accuracy of
with the NanoPhotometer. All validated libraries were mixed, Illumina short reads and the coverage of ONT long reads.
denatured, and diluted to the appropriate concentration for pair- Furthermore, Benchmarking Universal Single-Copy Orthologs
end 250-cycle sequencing on MiSeq. (BUSCO; v.4.0.0) (22) was used for genome completeness
For the GMNL-855 and BCRC-16100 strains, 1 µg DNA assessment through comparison with the lactobacillales_odb10
per sample was used as the input material for the DNA gene database, which contains 402 orthologs of 304 species
sample preparations. Sequencing libraries were generated using (23). The comparison of each chromosome with the reference
NEBNext Ultra DNA Library Prep Kit for Illumina (New England sequences was conducted using BLASTN and visualized with
Biolabs, Ipswich, MA, USA) following the manufacturer’s Easyfig (24).
recommendations, and index codes were added to attribute The genome was annotated through a multipronged approach
sequences to each sample. In brief, the DNA sample was consisting of the use of Prokka software (25) and a combination
fragmented through sonication to the size of 350 bp, and DNA of ab initio and evidence-driven gene prediction, including
fragments were then end-polished, A-tailed, and ligated using protein-coding regions and RNA genes (i.e., tRNA, rRNA). Then,
the full-length adaptor for Illumina sequencing with further PCR functional classification of these annotated genes was carried out
amplification. The PCR products were purified (AMPure XP); using eggNOG-mapper (26, 27) for annotation and classification
the libraries were then analyzed for size distribution by using in conjunction with the database of Clusters of Orthologous
an Agilent 2100 Bioanalyzer and quantified using real-time PCR. Groups (COGs) of proteins. The CRISPRFinder program was
The clustering of the index-coded samples was performed on a employed to search for CRISPR direct repeats and spacers
cBot Cluster Generation System according to the manufacturer’s (28). Mobile genetic elements such as conjugative plasmids,
Frontiers in Nutrition | www.frontiersin.org 4 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
bacteriophages, and transposons are regarded as a determinant (39) search tool v.2.2 in the Kyoto Encyclopedia of Genes and
force of horizontal gene transfer. Plasmid identification and Genomes (KEGG) database (https://www.kegg.jp/) was used for
typing were conducted using PlasFlow (29), and the genomes the investigation of undesirable genes.
were screened for prophages using the PHASTER tool (30);
transposon sequences were predicted using RepeatMasker RESULTS
software. Additionally, genes related to the production of
bacteriocins were predicted using BAGEL4 (31). Following Heat-Killed L. paracasei (GMNL-653)
annotation, the circular genome atlas was generated using the Reduced Levels of IL-6 and NO in
Circos visualization tool (32). LPS-Stimulated Mouse RAW 264.7
16S rRNA Phylogenetic Analysis Macrophages
To evaluate the quality of genome assembly, we compared The inflammation and bone cells are highly related. We used
the genome sequence of our strains with that of 12 reported the macrophage in vitro model to screen a variety of strains
Lacticaseibacillus strains from four L. paracasei species. The of heat-killed probiotics with anti-inflammatory activity, namely
publicly available 16S rRNA sequences of Lacticaseibacillus spp. L. reuteri, L. rhamnosus, L. salivarius, and L. paracasei. These
(L. acidophilus, L. amylovorus, L. casei, L. crispatus, L. delbrueckii, bacteria were inactivated at 90 ◦ C for 30 min and then added to
L. fermentum, L. gasseri, L. johnsonii, L. plantarum, L. reuteri, L. mouse RAW264.7 macrophages with or without LPS stimulation.
rhamnosus, and L. salivarius) were retrieved from the National Our results revealed that only L. salivarius GMNL-678 and
Center for Biotechnology Information nucleotide database L. paracasei GMNL-653 (Figure 1A, column 5 and 8) reduced
(https://www.ncbi.nlm.nih.gov/nuccore/?term=Lactobacillus). LPS-induced IL-6 production in the macrophages. The other
Then, the phylogenetic tree was constructed using Molecular Lactobacilli had no inhibitory effect on the production of IL-6
Evolutionary Genetics Analysis (MEGA X) software (33). In and NO in LPS-stimulated macrophages (Figure 1A). GMNL-
addition, the multiple sequence alignment program ClustalW 653 inhibited IL-6 and NO production under LPS stimulation in
was employed to reconstruct the molecular evolution tree a dose-dependent manner (Figures 1B,C).
using the maximum-likelihood estimation method, with genetic
distances computed using the Kimura two-parameter model. Heat-Killed GMNL-653 Increased Calcium
Confidence values for the phylogenetic trees were estimated Mineralization in Human Osteosarcoma
through bootstrap analyses with 1,000 replicates. MG63 Cells in Osteoinduction Medium
Bone is a mineralized tissue that continuously undergoes
Whole-Genome Average Nucleotide remodeling, with homeostasis achieved through the balance
Identity Analysis of osteoblasts and osteoclasts (40). First, we employed ARS
Although the 16S rRNA sequence-based division into higher taxa staining to evaluate calcium deposits using in vitro culture
is the most widely used classification system for prokaryotes, (41), demonstrating that a high dose (1 × 108 /mL) of
the sequence of 16S rRNA genes is too conservative to GMNL-653 markedly increased calcium deposits in the MG63
distinguish between closely related species. Hence, to measure the cells (Figure 2). Furthermore, we investigated whether heat-
nucleotide-level genomic similarity between our strain and other killed GMNL-653 triggered the differentiation of macrophages
related Lactobacillus genomes, the OrthoANI (34), regarded as into osteoclasts. Acid phosphatase-positive multinucleated (>3
the gold standard for prokaryotes at the genomic level, was nuclei) cells were counted as osteoclasts, revealing that GMNL-
calculated using the BLASTN program based on the modified 653 did not influence osteoclast differentiation under RANKL
algorithm of average nucleotide identity (ANI) (35). stimulation in vitro (Supplementary Figure 1).
Determination of Virulence Factors and GMNL-653 Protects Mice From
Antimicrobial Resistance Properties Ovariectomy-Induced Bone Loss
After genome annotation, each homolog of the L. paracasei To determine the effect of heat-killed probiotic GMNL-653
genes was searched in the BLAST database against the treatment on OVX-induced bone loss, 8-week-old ICR mice
Integrated Microbial Genomes and Microbiomes database were treated with a vehicle (H2 O), GMNL-653, and GMNL-
v.5.0 (36), Virulence Factor Database (VFDB) (37), and 678 once a day following OVX or sham surgery for 28 days.
Comprehensive Antibiotic Resistance Database (CARD) (38) Alendronate treatment acted as a positive control to treat and
to identify candidate virulence genes and antibiotic-resistant prevent osteoporosis in OVX mice (Table l). We performed high
genes using genotypic methods. The protein-coding gene was resolution micro-computed tomography for evaluation right
submitted into the Resistance Gene Identifier in the CARD for tibia bone morphology and architecture with treatment GMNL-
identification of antimicrobial resistance genes under the default 653 in OVX mice. The result showed that GMNL-653 enhances
settings (perfect/strict option). The BLAST hit cutoff values, bone microarchitecture in OVX mice (Supplementary Figure 2).
and an E < 1E-30 were used to identify the possible virulence In the vehicle-treated mice, OVX decreased BV/TV and
genes and antibiotic-resistant genes for further investigation of BMD, whereas the GMNL-653-treated mice exhibited increased
mobile genetic elements such as bacteriophages, plasmids, naked BV/TV and BMN. The effects of GMNL-678 were not as
DNA, or horizontal transposons. In addition, the BlastKOALA obvious as those of GMNL-653. Decreased mRNA levels of
Frontiers in Nutrition | www.frontiersin.org 5 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
FIGURE 1 | Heat-killed probiotics reduced IL-6 and NO levels through LPS stimulation in mouse RAW 264.7 macrophages. An appropriate number of cells were
seeded in a 24-well plate. Probiotics (L. reuteri, L. rhamnosus, L. salivarius, and five strains of L. paracasei) were cultured in MRS broth for 20 h, with the bacterial
concentration adjusted through centrifugation. These bacteria were heat killed (90◦ C for 30 min) and then added to cells with or without LPS stimulation. After 20 h,
the culture supernatant was collected for measurement of the IL-6 production (A,B) and NO (C). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the group
treated with LPS alone.
TGF-β and IL-10 and increased RANKL in the tibia were Relative abundance (Figure 4A) and diversity (Figure 4B)
observed in the OVX mice treated with H2 O (Figures 3A–C). of the fecal microbiota in the sham and OVX mice treated
However, GMNL-653 restored the TGF-β and IL-10 levels and with either the vehicle (H2 O) or GMNL-653 are depicted;
decreased the RANKL mRNA levels in the OVX mice. We also at the genus level, the dominant gut microbiota were rc4-4,
detect mRNA levels of bone morphogenetic proteins 2 (BMP2) Ruminococcus, Lachnospiracear, Mucispirillum, Oscillospira,
and tartrate-resistant acid phosphatase 5b (TRAP-5) in OVX Parabacteroides, Ruminococcaceae, Bacteroides, S24-7, and
mice (Supplementary Figure 3). BMP-2 plays a critical role in Clostridiales (Figure 4A). The results of the PCoA plot of beta
osteoblast differentiation (42). TRAP-5 has been used as a reliable diversity (Figure 4B) indicated that the microbial community,
biomarker of bone resorption and osteoclast number (43). There compared with the sham, OVX, and GMNL-653-treated OVX
has been an increasing trend in mRNA levels of BMP-2 in mice groups, exhibited a significantly distinct cluster that was
GMNL-653 treated OVX mice. The GMNL-653 decreased TRAP- separate from the other groups.
5 levels in OVX mice. In the OVX mice, IL-17A, and LPS levels in The differential microbiota from the three groups
the sera also increased, though GMNL-653 decreased these levels were identified through LEfSe analysis (Figure 5A), which
(Figures 3D,E). These data indicated that GMNL-653 protects demonstrated that Tenericutes (phylum) and Mollicutes (class)
mice from ovariectomy-induced bone loss and enhances gut were enriched in the OVX group and Lachnospiraceae (family),
barrier integrity. Rhizobiales (order), Aeromonadaceae (family), Bifidobacteriales
(order), Bifidobacterium (genus), Bifidobacteriaceae (family), and
GMNL-653 Modulated Gut Microbiota Rikenellaceae (family) were enriched in the OVX+GMNL-653
Composition in OVX Mice group. A PCA biplot was generated to investigate the microbial
Because gut barrier integrity may have been affected in the OVX composition differences between the sham, OXV, and OVX
mice, we further analyzed the gut microbiota compositions. + GMNL-653 groups (Figure 5B). This biplot revealed that
Frontiers in Nutrition | www.frontiersin.org 6 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
GMNL-653 Intervention Influenced the
Relative Abundance of the PICRUSt
Functional Prediction of Colonic
Microbiota in OVX Mice
To investigate the effect of GMNL-653 on the functional profiles
of microbiome communities in the OVX mice, we used PICRUSt
to predict metagenomes based on 16S rRNA marker gene
sequences and the KEGG database. The results revealed that
the relative abundance of several metabolic pathways associated
with pathways of cancer, pyruvate metabolism, carbohydrate
digestion and absorption, nitrogen metabolism, and geraniol
degradation in the colon of OVX mice were altered through
GMNL-653 treatment (Figure 6A). The relative abundance of the
functional pathways in carbohydrate digestion and absorption,
fatty acid biosynthesis, ion channels, nitrogen metabolism, and
other ion-coupled transporters as well as the pathways of
cancer, phenylalanine, tyrosine, and tryptophan biosynthesis, and
pyruvate metabolism were downregulated in the OXV group
compared with those in the sham group; they were, however,
upregulated through GMNL-653 treatment (Figure 6B).
FIGURE 2 | Heat-killed L. paracasei (GMNL-653) increased calcium
Correlation Between Gut Microbiota
mineralization in human osteosarcoma MG63 cells mineralizing in culture. (A) Composition and Sera Concentrations of
Human osteosarcoma MG63 cells are incubated with heat-killed GMNL-653 in
osteoinduction medium for 28 days. Following fixation, the cells are stained
IL-17 and LPS in OVX Mice
with Alizarin Red S to measure calcium deposits. (B) Quantification of Alizarin Our results indicated that sera IL-17 and LPS increased in
Red S stain by the absorbance (405 nm) of the red extracts according to the the OVA mice, implying that gut microbiota composition may
manufacturer’s instructions. ***P < 0.001 compared with the control group. be associated with immune responses to osteoporosis. Because
GMNL-653 intervention could reverse the dysbiosis of gut
microbiota, we further investigated the associations between gut
TABLE 1 | Heat-killed L. paracasei (GMNL-653) protect mice from
microbiota composition and IL-17 and LPS levels in the OVX
ovariectomy-induced bone loss.
mice. We then assessed which specific species were enriched
Control OVX + OVX + OVX + OVX + or depleted in the OVA mice and whether this correlated
H2 O GMNL-653 GMNL-678 alendronate with the cytokine and LPS concentrations. Using Spearman’s
correlation coefficient, we also investigated the correlation
BV/TV (%)
strength of the phylum counted in the OVX mice (Figure 7A),
42.12 ± 2.4** 30.9 ± 1.1 36.80 ± 1.6** 32.38 ± 0.8* 34.88 ± 0.9**
with the results demonstrating that the enriched Bifidobacteriales
BMD (g/cm3 )
(order), Rikenellaceae (family), Bifidobacteriaceae (family), and
0.502 ± 0.04** 0.344 ± 0.04 0.444 ± 0.043** 0.38 ± 0.027* 0.426 ± 0.02*
Bifidobacterium spp. in the OVX + GMNL653 group had
*P < 0.05 and **P < 0.01 compared with the OVX+H2 O group. significantly negative correlations with sera IL-17. Moreover, the
enriched Tenericutes (phylum) and Mollicutes (class) in the OVX
group exhibited positive correlations with sera LPS, whereas the
enriched Aeromonadacae (family) in the OVX + GMNL-653
dimension 1 and dimension 2 explained 51.6 and 20.7% of the group was negatively correlated with sera LPS (Figure 7B).
variation in gut microbiota composition, respectively. Obvious
intergroup distances tended to form distinct clusters between the General Genome Features and
OVX group and the other two groups, indicating dissimilar gut Comparative Genome Analysis
microbiota. These results suggested that dysbiosis occurred in Whole-genomic DNA–DNA hybridization (DDH) is the gold
the OVX mice, which was then modulated through GMNL-653 standard for bacterial species delineation (44). NGS-based
intervention, leading to a similar configuration to the sham genome sequencing has also been widely applied to the
control group. The relative abundance of gut bacteria indicated taxonomy of microorganisms and was successfully used to
that the enrichment of Tenericutes (phylum) and Mollicutes distinguish between L. casei and L. paracasei strains (45).
(class) in the OVX mice could be reduced through GMNL-653 However, these technologies are time intensive, costly, and
supplementation. Likewise, the decreased Bifidobacteriales difficult to use routinely in laboratories. Comparison of the
(order), Bifidobacteriaceae (family), and Rikenellaceae (family) DNA sequences of protein-encoding genes is an alternative
abundance in the OVX mice was enriched by GMNL-653 approach to the analysis of whole-genome relatedness and
(Figure 5C). has become increasingly critical in the use of these sequences
Frontiers in Nutrition | www.frontiersin.org 7 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis FIGURE 3 | (A–C) The mRNA levels of TGF-α, RANKL, and IL-10 in the tibia. (D,E) The IL-17A and LPS levels in the sera of ovariectomized (OVX) mice treated with either the vehicle (H2 O) or heat-killed L. paracasei (GMNL-653). qRT-PCR analysis of the expression of genes known to promote bone formation, TGF-β and IL-10 (A,C), and bone resorption, RANKL, in the tibia of OVX mice. The levels of IL-17A and LPS in the sera are measured through ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the OVX+H2 O group. as molecular targets for microbial species identification. We Three L. paracasei strains (GMNL-653, GMNL-855, and identified and verified the distinct nature of the three strains of L. BCRC-16100) were characterized at the genotypic and paracasei (GMNL-653, GMNL-855, and BCRC-16100) by using phenotypic level (Figure 8). High-molecular-weight DNA SEM, RAPD analysis, and carbohydrate fermentation features from three isolates of the L. paracasei strain was sequenced using (Supplementary Figure 4). Furthermore, we performed genome ONT long reads and Illumina short reads for the hybrid assembly sequencing and characterization of the strains to identify genes of their genomes using the MaSuRCA assembler v3.3.1 (20). As potentially involved in probiotic activity. detailed in Table 2, the assembly of the GMNL-653 draft genome Frontiers in Nutrition | www.frontiersin.org 8 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis FIGURE 4 | Relative abundance and diversity of stool microbiota in the sham and OVX mice treated with either the vehicle (H2 O) or heat-killed GMNL-653. (A) Each column in the stacked histogram represents one sample; the differently colored bars are proportional to the percentage of relative bacterial abundance of each taxon within the sample (summing up to 100%). (B) Principal coordinate analysis of the beta diversity among the three groups. Frontiers in Nutrition | www.frontiersin.org 9 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis FIGURE 5 | Taxonomic differences and relative abundance of fecal microbiota in the sham and OVX mice treated with either the vehicle (H2 O) or heat-killed GMNL-653. (A) Linear discriminant analysis and cladogram revealed the phylogenetic distribution of fecal microbiota associated with the sham, OVX, and OVX + GMNL-653 groups. The biplot of the principal component analysis (B) and relative abundances (C) of bacterial communities in the sham, OVX, and OVX+GMNL-653 groups. Frontiers in Nutrition | www.frontiersin.org 10 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis FIGURE 6 | Predicted function of the involved gut microbial using the phylogenetic investigation of communities by the reconstruction of unobserved states 2 (PICRUSt2) in the sham and OVX mice treated with either the vehicle (H2 O) or heat-killed GMNL-653. (A) Pairwise comparison showed that five function pathways were significantly increased in mice treated with GMNL-653 vs. OVX mice. Among them, 4 of 5 function pathways were included in the metabolism of pyruvate, carbohydrate, nitrogen, and geraniol. (B) There were nine function pathways overlapped between OVX vs. GMNL-653 and Sham vs. OVX and were significantly decreased in the OVX mice as compared to the Sham group, but further increased upon GMNL-653 treatment. It is noted that only Lysine degradation pathway was overlapping between OVX vs. GMNL-653 and Sham vs. GMNL-653 and was significantly increased in the OVX mice as compared to the Sham group, but was further increased when treated with GMNL-653. p < 0.05 is considered as statistically significant. Frontiers in Nutrition | www.frontiersin.org 11 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis FIGURE 7 | Correlations between the gut microbiota composition and plasma concentrations of IL-17 and LPS in the sham and OVX mice treated with either the vehicle (H2 O) or heat-killed GMNL-653. (A) Spearman’s correlation coefficient strength of the phylum counts from the OVX mice. Correlations with P < 0.05 are indicated, and the strength of the coefficient is represented by the change in color intensity. (B) The enriched Bifidobacteriales (order), Rikenellaceae (family), Bifidobacteriaceae (family), and Bifidobacterium spp. in the OVX + GMNL653 group exhibited a significant negative correlation with sera IL-17. (C) The enriched Tenericutes (phylum) and Mollicutes (class) in the OVX group were positively correlated with sera LPS. The enriched Aeromonadacae (family) in the OVX + GMNL-653 group exhibited a negative correlation with sera LPS. sequence consists of three contigs amounting to 3,329,730 bp. (32). For completeness assessment for genomics data quality The first contigs (3,121,847 bp) were regarded as a circular control, BUSCO analysis was employed, revealing that 400 of chromosome predicted using PlasFlow (29), with a GC content the 402 reference genes were complete (99.5%, 391 single-copy of 46.19%. Prokka (25) software was used to annotate 3,375 and 9 duplicated), 2 (0.5%) were fragmented, and none were protein-coding genes and identify 60 tRNA genes and five rRNA missing in the draft assembly; thus, the assembly represents most operons. The genetic organization of GMNL-653 was illustrated of the genomic content (Supplementary Figure 5). One separate as a genome atlas constructed with Circos software (Figure 8A) CRISPR loci (Table 2, Supplementary Table 1) was discovered Frontiers in Nutrition | www.frontiersin.org 12 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis FIGURE 8 | Circular genome graph (A–C) of the L. paracasei strain. (A) The GMNL-653, (B) GMNL-855, and (C) BCRC-16100 genome. The outer to inner circles depict the (1) DNA coordinates; (2) function-based color-coded mapping of the genes, colored according to the COG category whose code and meaning is provided in the table below the plot; (3, 4) coding sequences predicted on the forward (red) and reverse (blue) strands, respectively; (5) GC skew (G – C/G + C), calculated for a window of 4,000 bp and shifted by 1,000 bp over the genome sequence, with the above- and below-average regions indicated in green and purple; (6) GC content (black); and the (7) RNA genes (tRNA, blue; rRNA, red; other RNAs, black). in the genome of GMNL-653 by using the CRISPRFinder The complete genome sequence of GMNL-855 and BCRC- (28). Additionally, eight prophage regions were identified, 16100 comprised a circular chromosome (3,118,787 and of which four regions were intact, two were incomplete, and 3,029,023 in contigs 1, respectively), with average GC values two were questionable (Supplementary Table 2); a type IIa of 46.4 and 46.19%, respectively. Figures 8B,C illustrates the bacteriocin operon (Supplementary Table 3) was also identified orthologous genes shared among strains, presenting the position in GMNL-653. and color-coded function of each specific gene. Through BUSCO Frontiers in Nutrition | www.frontiersin.org 13 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
TABLE 2 | General features of L. paracasei genomes. the diagonals departing from each strain. The critical ANI
value aligned to other species was between 64.94 and 79.55%.
GMNL-653 GMNL-855 BCRC 16100
A BLAST comparison of GMNL-653 against the GMNL-
Size (bp) 3,329,730 3,307,405 3,029,023 855 and BCRC-16100 strains was performed using Easyfig
G + C content (%) 46.19 46.19 46.4 (Supplementary Figure 8). Genome-wide comparison indicated
Total genes 3,375 3,328 2,902 a high degree of synteny among the characterized L. paracasei
Coding content (%) 88.82 86.27 85.88
GMNL-653, GMNL-855, and BCRC-16100 strains, with only a
Gene average length (bp) 852 857 896
few regions disrupted throughout the chromosome. Therefore,
Genes assigned to COGs 2,627 (77.8%) 2,669 (80.2%) 2,387 (82.2%)
the whole-genome data verified that the GMNL-653 strain
Chromosome 1 1 1
belongs to the species L. paracasei. The functional classification
of Clusters of Orthologous Groups (COGs) predicted using
rRNA operons 5 5 5
eggNOG-mapper (26, 27) revealed that 2,627 (77.8%) putative
tRNA 60 60 62
coding sequences were homologous to known gene families.
Plasmids 0 0 0
The distribution of genes into COG functional categories is
Transposases 16 104 13
summarized in Table 3. Functional analysis of GMNL-653 genes
CRISPR loci 1 1 2
revealed that, in addition to hypothetical proteins, a relative
Prophage-Like clusters 4 5 0
abundance of the gene was involved in the basic mechanisms of
Bacteriocin 3 3 2
DNA replication and recombination, carbohydrate metabolism,
and cell wall/membrane/envelope biogenesis. Additionally, in the
putative regions of genomic coding, 2,669 (80.2%) and 2,387
(82.2%) genes were attributed to a COG family (Table 3). A core-
analysis, we determined that 99.5% of reference genes were
and pan-genome analysis indicated that 72, 40, and 34 genes
complete in both strains, 2 (0.5%) were fragmented, and none
were specific to GMNL-653, GMNL-855, and BCRC-16100,
were missing (Supplementary Figure 5). In total, over 99%
respectively (Supplementary Table 4).
of complete BUSCOs were present in the three L. paracasei
Cell wall components are key mediators influence on
strains, indicating high-quality assemblies. Of the 3,328 and
the interactions between host and associated microbiota.
2,902 genes that were predicted in GMNL-855 and BCRC-
Lacticaseibacillus not only utilize and metabolize a variety of
16100, respectively, 86.27 and 85.88% were protein-coding genes,
carbohydrates but also synthesized extensive exopolysaccharides
respectively. Gene prediction demonstrated the presence of the
which may affect other commensal bacteria resulting in health
following putative genes: three CRISPR/CRISPR-associated (cas)
benefits in host. Therefore, genome sequencing of many
gene loci (type I and IIA; Supplementary Table 1), five intact
Lactobacillus strains have revealed many genes involved in
prophage-like regions (Supplementary Table 2), and several type
the metabolism of carbohydrate. Based on our results, we
IIb bacteriocin operons (Supplementary Table 3).
proposed that the bacterial cell wall components or carbohydrate
metabolism exclusively expressing in GMNL-653 may be
Genotypic Identification of the L. paracasei involved in the antiosteoporotic activity. We compared genes
Strain related to carbohydrate transport and metabolism (Figure 9A)
The beneficial health attributes and safety concerns of probiotic and cell wall/membrane/envelope biogenesis (Figure 9B) among
cultures are closely related; thus, categorical identification of the three L. paracasei strains. Thirteen genes were annotated to
the probiotic strain is paramount (46). A maximum-likelihood carbohydrate transport and metabolism, which are sucrose-6-
tree of four L. paracasei genomes and 12 reported Lactobacillus phosphate hydrolase, phosphotransferase (PTS) system, glycerate
strains (L. acidophilus, L. amylovorus, L. casei, L. crispatus, L. kinase, sucrose phosphorylase, phosphotransfer between the C1
delbrueckii, L. fermentum, L. gasseri, L. johnsonii, L. plantarum, and C5 carbon atoms of pentose, glycoside hydrolase family
L. reuteri, L. rhamnosus, and L. salivarius) was created based on 4, repressor, ORF, kinase (ROK) family, glycosyl hydrolase
16S rRNA gene sequences. Genome-wide phylogenetic analysis family 1, and sugar kinase (Table 4). Six genes were related
in combination with alignment fraction analysis indicated to cell wall/membrane/envelope biogenesis, which are phage
that GMNL-653, GMNL-855, BCRC-16100, and L. casei spp. tail tape measure protein, udp-galactopyranose mutase, glycosyl
were grouped together (Supplementary Figure 6). Another work transferase, licD family, and catalyzes the conversion of a
documented an approximately 90% sequence identity among range of fructosamine 6-phosphates to glucose 6phosphate,
L. casei, L. paracasei, and L. rhamnosus (47). Furthermore, and a free amino acid (Table 4). According to the KEGG
to measure genomic similarity at the nucleotide level between BRITE database, these thirteen genes annotated to carbohydrate
GMNL-653 and other Lactobacillus genomes, the average transport and metabolism have functional hierarchy classification
nucleotide identity (ANI) was calculated using an improved of starch and sucrose metabolism, fructose-specific II-like
algorithm (34). The ANI values were calculated and revealed component, biosynthesis of secondary metabolism, pentose
that three L. paracasei strains were most closely related to phosphate pathway/purine metabolism, mitochondrial DNA
L. paracasei LC2W in terms of their nucleotide sequences transcription and translation factors, fructose like PTS system
(ANI > 98%; Supplementary Figure 7). The values between EIIA component, and fructoselysine 6-kinase. Six genes related
the two strains were extracted from the junction point of to cell wall/membrane/envelope biogenesis have functional
Frontiers in Nutrition | www.frontiersin.org 14 February 2022 | Volume 9 | Article 804210Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
TABLE 3 | COG functional categories of L. paracasei strain.
COG class Description GMNL-653 GMNL-855 BCRC 16100
J Translation, ribosomal structure, and biogenesis 107 (4.1%) 157 (5.9%) 149 (6.2%)
L Replication, recombination, and repair 304 (11.6%) 258 (9.7%) 135 (5.7%)
K Transcription 209 (8.0%) 212 (8.0%) 201 (8.4%)
D Cell cycle control, cell division, chromosome partitioning 29 (1.1%) 28 (1.1%) 25 (1.0%)
V Defense mechanisms 104 (4.0%) 103 (3.9%) 81 (3.4%)
T Signal transduction mechanisms 60 (2.3%) 62 (2.3%) 64 (2.7%)
M Cell wall/membrane/envelope biogenesis 129 (4.9%) 136 (5.1%) 138 (5.8%)
N Cell motility 2 (0.1%) 1 (0.1%) 1 (0.1%)
U Intracellular trafficking, secretion, and vesicular transport 20 (0.8%) 24 (0.9%) 16 (0.7%)
O Post-translational modification, protein turnover, and chaperones 71 (2.7%) 65 (2.4%) 61 (2.6%)
C Energy production and conversion 107 (4.1%) 113 (4.2%) 110 (4.6%)
G Carbohydrate transport and metabolism 270 (10.3%) 314 (11.8%) 307 (12.9%)
E Amino acid transport and metabolism 207 (7.9%) 203 (7.6%) 196 (8.2%)
F Nucleotide transport and metabolism 88 (3.3%) 88 (3.3%) 87 (3.6%)
H Coenzyme transport and metabolism 47 (1.8%) 46 (1.7%) 47 (2.0%)
I Lipid transport and metabolism 55 (2.1%) 54 (2.0%) 52 (2.2%)
P Inorganic ion transport and metabolism 113 (4.3%) 113 (4.2%) 114 (4.8%)
Q Secondary metabolites biosynthesis, transport, and catabolism 18 (0.7%) 13 (0.5%) 16 (0.7%)
S Function unknown 687 (26.2%) 676 (25.4%) 587 (24.6%)
hierarchy classification of glycan biosynthesis and metabolism, diseases, and non-alcoholic fatty liver disease. Disruption of
lipopolysaccharide cholinephosphotransferase and fructoselysine gut microbiota, also called dysbiosis, is also related to multiple
6-phosphate deglycase. host disorders and is closely associated with an increased risk
of bone loss (48), inflammatory bowel disease (49), diabetes,
and obesity (50). Although the potential causality between
DISCUSSION various microbial components and diseases must be clarified,
the intervention of probiotics or prebiotics can still provide
In present study, we observed that one L. paracasei GMNL-653 beneficial metabolic health effects. In this study, we applied
strain reduced LPS-stimulated IL-6 and NO production in the in vitro and in vivo models to validate the antiosteoporotic
mouse macrophage RAW264.7. GMNL-653 increased calcium effect of heat-killed L. paracasei (GMNL-653) and employed
mineralization in human osteosarcoma MG63 cells mineralizing comparative genomics analysis to explore the potentially vital
in culture and protected the OVX mice from bone loss, including genes involved.
increasing bone volume over tissue volume (BV/TV) and bone Osteoporosis is mediated by a variety of inflammatory
mineral density (BMD). Our results revealed that GMNL- mediators, including TNF-α, RANKL, interleukin 1 beta, IL-
653 restored ovariectomy-induced gut microbiota dysbiosis 6, and interferon gamma. Furthermore, chronic inflammatory
and maintained intestinal barrier integrity. Additionally, in disease such as inflammatory bowel disease can affect bone
the OVX mice, GMNL-653 treatment reduced the IL-17 metabolism and is frequently associated with occurrence of
and LPS levels compared with those of the vehicle control osteoporosis (5, 7, 51). Although our understanding of the
group, reduced the mRNA levels of RANKL, and increased underlying mechanism of the relationship between inflammation
the anti-inflammatory cytokines TGF-β and IL-10 in tibia and osteoporosis is incomplete, increasing evidence continues
tissue. Furthermore, we applied the whole-genome sequencing demonstrated that inflammation may intrinsically contribute to
technique and comparative genomics analysis to further examine osteoporosis. Based on these findings indicating the contribution
these three L. paracasei. Our results indicated that the genes of inflammation to bone loss, we screened the anti-inflammatory
related to carbohydrate transport/metabolism and the cell activity in various strains of heat-killed probiotics in RAW
wall/membrane/envelope biogenesis of GMNL-653 are worthy of 264.7 macrophages under LPS stimulation. We observed that
future investigation. In summary, our results showed that heat- one heat-killed L. paracasei GMNL-653 reduced macrophage
killed GMNL-653 exhibited antiosteoporotic activity through the IL-6 secretion and NO production following LPS treatment.
gut microbiota–bone axis (Supplementary Figure 9). RANKL is a key regulator of osteoclast activity and acts as a
Many studies have explored the potential role of the intestinal therapeutic target in osteoporosis (52). Our results indicated that
microbiome and its associated metabolomics in metabolic health the levels of RANKL mRNA increased in OVX mice, though
and disease. Several metabolic diseases have been linked to subsequent treatment with heat-killed GMNL-653 once per day
altered gut microbiota, such as type 2 diabetes, cardiometabolic resulted in decreased RANKL mRNA expression in the tibia.
Frontiers in Nutrition | www.frontiersin.org 15 February 2022 | Volume 9 | Article 804210Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis FIGURE 9 | Comparison of the genes related to (A) carbohydrate transport and metabolism and (B) cell wall/membrane/envelope biogenesis among the three L. paracasei strains. Furthermore, GMNL-653 increased TGF-β and IL-10 mRNA IL-17A is also a vital mediator of bone absorption in levels in the OVX mice. These results demonstrated that GMNL- inflammatory diseases associated with osteoporosis, and elevated 653 exhibits anti-inflammatory effects, with anti-inflammatory serum concentrations of IL-17A have been observed in cytokines potentially enhancing gut epithelial barrier function patients with postmenopausal osteoporosis (56). We determined (53, 54) and proinflammatory cytokines attenuating intestinal that elevated IL-17A was downregulated through GMNL-653 barrier dysfunction (55). treatment in OVX mice and that IL-17 can also promote Frontiers in Nutrition | www.frontiersin.org 16 February 2022 | Volume 9 | Article 804210
Jhong et al. Lacticaseibacillus paracasei Regulate Gut–Bone Axis
TABLE 4 | EggNOG functional annotations of L. paracasei (GMNL-653). osteoclast differentiation under RANKL stimulation. These
results demonstrated that GMNL-653 can promote osteoblast
Functional Predicted eggNOG annot KEGG brite
categories gene name
differentiation leading to mineralization. The use of the OVX
mice model verified that the GMNL-653 protects mice from
Carbohydrate SACC Sucrose-6-phosphate Starch and sucrose ovariectomy-induced bone loss.
transport and hydrolase metabolism In the OVX mice, gut integrity was also altered, leading to
metabolism elevated LPS in the sera. Notably, the gut microbiota composition
(G)
changed in the OVX group. The PCoA plot of our beta
FRYC PTS System Fructose-specific II-like
component
diversity analysis indicated that the microbial communities in
the OVX mice exhibited a significantly distinct cluster separate
GLXK Glycerate kinase Biosynthesis of
secondary metabolites from the sham and OVX + GMNL-653 groups. Accumulated
AMYA Sucrose phosphorylase Starch and sucrose evidence revealed that the gut microbiome was associated with
metabolism specific dysbiosis in osteoporosis, though the causal mechanism
YHFW Phosphotransfer Pentose phosphate remains unclear. Epidemiological study also reported that the
between the C1 and pathway/Purine gut microbiome was altered in postmenopausal women with
C5 carbon atoms of metabolism osteoporosis and osteopenia (58). A potential mechanism is that
pentose (By similarity)
dysbiosis of gut microbiota may increase the permeability of the
MALH Glycoside hydrolase Starch and sucrose
intestinal cell, increasing LPS levels in the circulation system.
family 4 metabolism
LPS can upregulate the inflammatory mediators in bone tissue
SP_2163 PTS system
(59). LPS has identified as the vital factor in inflammatory-
GATC PTS system, Mitochondrial DNA
galactitol-specific IIc transcription and
induced osteoclast differentiation leading to bone loss (60, 61).
component translation factors Levels of sera IL-17A and LPS correlated with certain gut
SP_2142 ROK family microorganisms in the OVX mice, indicating a link between
GMUD Glycosyl hydrolase Starch and sucrose immune responses and changes in gut microbiome composition.
family 1 metabolism In addition, gut microbiota may be involved in the regulation of
FRVA PTS system Fructose-like PTS IL-17A production and functions (62). Therefore, we speculated
system EIIA component that GMNL influences gut microbiota, leading to decreased IL-
MANP PTS system 17A and LPS production in OVX mice.
FRLD Sugar kinase Fructoselysine 6-kinase The enriched Bifidobacteriales (order), Rikenellaceae (family),
Cell wall/ ELI_1307 Phage tail tape Bifidobacteriaceae (family), and Bifidobacterium spp. in the
membrane/ measure protein OVX + GMNL653 group exhibited significantly negative
envelope
correlations with sera IL-17A. GMNL-653 intervention can
biogenesis
(M) enhance the abundance of these microorganisms, resulting in
GLF udp-galactopyranose Glycan biosynthesis decreased IL-17A production. Our results demonstrated that
mutase and metabolism GMNL-653 modulated gut microbiota composition in OVX
RFAB Glycosyl transferase Glycan biosynthesis mice. This result implied that the interactions of Rikenellaceae
(Group 1 family protein) and metabolism (family), Bifidobacteriales (order), Bifidobacteriaceae (family),
LSGF Glycosy ltransferase Bifidobacterium spp. may be highly associated with IL-17.
LICD3 licD family lipopolysaccharide These subset of gut microbiota may have selective effects on
cholinephosphotransferase IL-17. Previous study reported that Bifidobacterium suppress
FRLB Catalyzes the fructoselysine IL-17 production in murine splenocytes and dextran sodium
conversion of a range 6-phosphate deglycase
sulfate-induced intestinal inflammation (63). However, whether
of fructosamine
6-phosphates to
the alterations in the abundances of Bifidobacterium and
glucose 6-phosphate Rikenellaceae contribute to the functional consequence on the
and a free amino acid homeostasis of osteoblast and osteoclast development are issues
(By similarity) that need further clarified. Our finding is consistent with a study
reporting that gut Rikenellaceae involved in Th17 development
(64). The enriched Tenericutes (phylum) and Mollicutes (class) in
OVX group have positive correlations with sera LPS. Tenericutes
osteoclast differentiation and RANKL secretion. Our results has been reported as one of biomarkers in rat model of senile
indicated that elevated IL-17 may enhance RANKL expression osteoporosis (65). Our result indicated that enriched Tenericutes
in OVX mice. Blocking the IL-17A pathway may have a was highly associated with gut barrier integrity which may
positive effect on bone and cartilage damage in inflammatory result from intestinal inflammations. We further investigated the
disease (57). Furthermore, GMNL-653 may reduce IL-17A effect of GMNL-653 on the functional profiles of microbiome
production, preventing bone loss in OVX mice. In terms communities in the OVX mice and used PICRUSt to predict
of the influence of GMNL-653 osteoblast and osteoclast metagenomes mapped to the KEGG database. Carbohydrate
differentiation, we discovered that GMNL-653 does not influence digestion and absorption, fatty acid biosynthesis, ion channels,
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